New technique speeds identification of potential drug candidates

RICHLAND, Wash. —
Looking for compounds that have the right stuff to become new drugs can be like trying to find a needle in a haystack. But a new use of mass spectrometry could benefit the pharmaceutical industry by making it faster and easier to identify potential drugs.

Harvard University has confirmed results of early tests of a new technique being developed by researchers at the Department of Energy's Pacific Northwest National Laboratory.

Researchers at the laboratory are using one-of-a-kind instruments to determine how well groups of molecules bind to a specific target molecule. Strong binding is one indication a compound may be a useful drug. The bioaffinity characterization technique developed at Pacific Northwest allows a large number of related compounds to be screened for specific binding activity and simultaneously identified -- significantly decreasing drug lead discovery time.

In recent years, pharmaceutical companies have used combinatorial chemistry to rapidly combine chemical compounds to create "libraries" that can contain thousands of even millions of related compounds. While this often can be done rapidly, screening the libraries for potentially useful characteristics takes much longer. Current techniques also can give misleading results or miss a lead altogether, researchers say.

When looking for new drugs to fight illness, drug researchers often identify enzymes that control a specific reaction. For example, victims of ethylene glycol (antifreeze) poisoning typically are administered a nearly intoxicating dose of ethanol (alcohol) because the enzyme that converts ethylene glycol to a toxic oxidation product is effectively inhibited or turned off by ethanol. Thus, ethanol blocks the conversion of ethylene glycol to a toxic form and allows it to be excreted harmlessly. Enzymes can be thought of as a lock with the potential inhibitor serving as a key that fits the lock and binds tightly to the enzyme.

"To understand how these inhibitor molecules target an enzyme, ideally, you would put them in solution and look at the interaction," said George Whitesides, professor of chemistry at Harvard University. "So, why haven't people done that? Because there hasn't been any way of analyzing that interaction."

Now there is, thanks to the use of electrospray ionization combined with advanced mass spectrometric methods, a technique pioneered by Richard Smith and his colleagues at Pacific Northwest. Until recently, it could not be done because heating the solution to convert it to the vapor state for analysis in a mass spectrometer destroys the molecules. ESI, however, does not use heat but creates a fine spray of highly charged droplets which evaporate, leaving intact molecules ready for analysis by powerful new mass spectrometers. The technique even maintains the fragile complexes of enzymes with inhibitors. "The ability to put large molecules in a vapor state is remarkable," Whitesides said.

Smith and researchers at Pacific Northwest's new Environmental and Molecular Sciences Laboratory inject the molecules as ions into a Fourier transform ion cyclotron resonance spectrometer, which measures their masses with far greater accuracy and power than any other type of mass spectrometer. This led to the discovery that, under proper experimental conditions, the abundance of a specific inhibitor can give information about its binding strength. The technique has worked on a library of 289 inhibitors. EMSL researchers selected the tightest binding inhibitors, and the Harvard team confirmed the finding using conventional techniques.

"Inhibitors are similar to vehicles competing for parking places at the mall," explained Steve Hofstadler, an EMSL researcher. "If you have several mini-vans, sedans and sports cars competing, and the prime parking places are quite small, only one make of the sports cars might be able to fit in." When inhibitors are competing, the best binding ones, giving the best "fit," will occupy more of the possible binding sites. The identity and the number of winners in this competition can be measured with mass spectrometry.

The EMSL process is very fast. Each experiment takes only a minute or two. Researchers estimate that as the method is refined, tens of thousands to many millions of compounds could be analyzed for high binding species and their relative binding affinities in one afternoon -- an undertaking which might require weeks using conventional screening methods. Researchers add that even if this does not result directly in the discovery of a new drug, it can quickly rule out unpromising compounds and help drug developers focus on more promising leads. It also can provide important fundamental insights into the nature of the binding site.

"Right now, armies of post doctoral candidates and graduate students are used in biological research to accomplish the same thing," said Smith. "This is a way to reduce by several orders of magnitude, in some cases, their effort in getting to solutions."

EMSL researchers are continuing to develop the techniques using actual drug candidates provided by several pharmaceutical companies. Smith and his team also plan to apply this powerful technique to a fascinating environmental and health issue. They will study how the body's mechanism for repairing DNA, damaged by chemicals or radiation, sometimes goes awry. The results may help in the search for cures to diseases such as cancer.

Interdisciplinary teams at Pacific Northwest National Laboratory address many of America's most pressing issues in energy, the environment and national security through advances in basic and applied science. Founded in 1965, PNNL employs 4,400 staff and has an annual budget of nearly $1 billion. It is managed and operated by Battelle for the U.S. Department of Energy's Office of Science. As the single largest supporter of basic research in the physical sciences in the United States, the Office of Science is working to address some of the most pressing challenges of our time. For more information on PNNL, visit the PNNL News Center, or follow PNNL on Facebook, Google+, Instagram, LinkedIn and Twitter.